KR20130095454A - Method for distinguishing between mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor - Google Patents
Method for distinguishing between mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor Download PDFInfo
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Abstract
Description
본 발명은 결핵균 및 비결핵 항산균의 구별방법, 그 방법에 사용되는 조성물 및 키트에 관한 것이다.The present invention relates to a method for distinguishing Mycobacterium tuberculosis and non-tuberculosis antibacterial bacteria, compositions and kits used in the method.
일반적으로 결핵은 결핵균(Mycobacterium tuberculosis, MTB)이 원인이 되어 발생하는 만성 감염성 질환이고 우리나라에서 결핵의 유병률은 지속적으로 감소하는 추세이나 외국에 비해 높은 편이다[World Health Organization. Global tuberculosis control: surveillance, planning, financing (WHO Report 2004). Geneva: World Health Organization, 2004]. 또한, 효과적인 항결핵제가 사용되어 왔음에도 불구하고 아직도 세계적으로 결핵환자가 매년 800만 명이 발병하고 결핵으로 인해서 200만 명이 사망하고 있다[Chang HE, Heo SR, Yoo KC, Song SH, Kim SH, Kim HB, et al. Detection of Mycobacterium tuberculosis complex using Real-time Polymerase chain reaction. Korean J Lab Med 2008;28:103-8]. In general, tuberculosis is Mycobacterium tuberculosis (MTB) is a chronic infectious disease caused by tuberculosis, and the prevalence of tuberculosis in Korea continues to decrease, but is higher than in other countries [World Health Organization. Global tuberculosis control: surveillance, planning, financing (WHO Report 2004). Geneva: World Health Organization, 2004]. In addition, despite the effective use of anti-tuberculosis drugs, there are still 8 million cases of tuberculosis in the world each year and 2 million people die from tuberculosis [Chang HE, Heo SR, Yoo KC, Song SH, Kim SH, Kim HB]. , et al. Detection of Mycobacterium tuberculosis complex using Real-time Polymerase chain reaction. Korean J Lab Med 2008; 28: 103-8.
결핵의 원인균으로는 결핵균뿐만 아니라 외국의 경우 비결핵 항산균(nontuberculous mycobacteria, NTM)의 빈도가 높은 지역에서는 항산균 도말 양성 객담의 30%에서 50%까지 비결핵 항산균이 분리되고 있으며 선진국화될수록 비결핵 항산균에 의한 결핵환자의 발생이 증가하는 것으로 보고되면서 마이코박테리아 균동정이 매우 중요한 문제가 되고 있다[Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998;36:1046-9;Marras TK and Daley CL. Clin Chest Med 2002;23:553-67; This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000;55:210-8]. The most common causes of tuberculosis are tuberculosis bacteria, as well as nontuberculous mycobacteria (NTM) in foreign countries, where non-TB bacteria are isolated from 30% to 50% of antiseptic smear-positive sputum. Mycobacterial bacteriological identification has become a very important issue as it is reported that the incidence of tuberculosis patients due to non-tuberculosis acid bacillus is increased [Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998; 36: 1046-9; Marras TK and Daley CL. Clin Chest Med 2002; 23: 553-67; This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000; 55: 210-8].
국내의 경우 비결핵 항산균 질환의 빈도가 낮다고 알려져 있었기 때문에 항산균 도말양성일 경우 대부분 결핵으로 간주하고 항결핵제 치료를 시행하는 것이 일반적이었으나, 국내에서도 항산균 도말 양성 객담의 10.3-12.2%까지 비결핵 항산균의 검출이 보고되어 왔으나 최근에는 30% 수준까지 그 비율이 높아지고 있는 추세이다[Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000;55:210-8;Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Tuberc Respir Dis 2003;54:22-32]. In Korea, it was known that the incidence of non-TB disease was low, so it was generally considered that tuberculosis was considered to be tuberculosis, and anti-TB treatment was generally performed. Bacterial detection has been reported, but the rate has recently increased to 30% [Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000; 55: 210-8; Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Tuberc Respir Dis 2003; 54: 22-32.
또한 비결핵 항산균은 면역 기능 저하자에서 질병을 일으킬 수 있고 진단이 쉽지 않으며 균종에 따라 자연적으로 가지는 약재내성이 다르므로 비결핵 항산균에 의한 결핵의 경우 그 치료법이 결핵균에 의한 결핵의 치료법과 다를 수 있다[Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000;55:210-8;Koh WJ, Kwon OJ, Lee KS. a Korean perspective. J Korean Med Sci 2005;20:913-25;Wagner D and Young LS. Infection 2004;32:257-70]. 그러므로 결핵균과 비결핵 항산균을 신속하게 구별할 수 있는 적절한 검사방법이 요구되고 있다.In addition, since non-TB tuberculosis can cause disease in people with reduced immune function, it is not easy to diagnose, and the drug resistance naturally differs depending on the type of bacteria. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000; 55: 210-8; Koh WJ, Kwon OJ, Lee KS. a Korean perspective. J Korean Med Sci 2005; 20: 913-25; Wagner D and Young LS. Infection 2004; 32: 257-70. Therefore, there is a need for an appropriate test method that can quickly distinguish between tuberculosis bacteria and non-TB bacteria.
결핵의 진단에는 보편적으로 항산균 도말검사와 배양검사를 이용해왔다. 항산균 도말검사는 신속하게 결과를 얻을 수 있고 기법이 간편하다는 장점이 있으나 민감도와 특이도가 떨어지며 결핵균과 비결핵 항산균을 구분할 수 없다는 단점이 있으며 배양검사는 결핵진단의 표준법으로 결핵을 진단하는 가장 정확한 방법이지만 천천히 자라는 결핵균의 특성상 시간이 오래 걸린다는 단점이 있으며, 장기간 배양을 하기 때문에 다른 균에 의해 오염이 되는 경우가 많다[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8]. 최근 결핵균 배양시간을 단축하기 위해서 액체배지를 이용한 자동화 검사법이 등장하였으나 이 방법 역시 결핵균과 비결핵 항산균을 구별할 수 없기 때문에 별도의 동정과정이 필요하다는 단점이 있다[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8].Tuberculosis has been commonly used for the diagnosis of anti-bacterial smear and culture. The antimicrobial smear test has the advantages of rapid results and simple techniques, but it has the disadvantage that sensitivity and specificity are inferior, and that it is impossible to distinguish between tuberculosis bacteria and non-tuberculosis antibacterial bacteria. Although it is the most accurate method, it takes a long time due to the slow growth of Mycobacterium tuberculosis, and because it is cultured for a long time, it is often contaminated by other bacteria [Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8. Recently, automated test method using liquid medium has been introduced to shorten tuberculosis incubation time, but this method also has a disadvantage of requiring a separate identification process because it cannot distinguish between tuberculosis and non-tuberculosis antibacterial bacteria [Yang HY, Lee HJ, Park. WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8.
최근에는 분자진단 기술이 발달되면서 이를 이용한 결핵 진단법들이 많이 개발되었다[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8;Chakravorty S and Tyagi JS. J Clin Microbiol 2005;43:2697-702;Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008;28:34-8]. Recently, with the development of molecular diagnostic technology, tuberculosis diagnostic methods using the same have been developed [Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8; Chakravorty S and Tyagi JS. J Clin Microbiol 2005; 43: 2697-702; Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008; 28: 34-8.
결핵의 확실한 진단방법은 결핵균에 의한 감염을 증명하는 것이므로 임상 검체에서 직접 MTB를 검출하는 방법이 가장 확실하다. 따라서 PCR, real-time PCR, hybridization, oligonucleotide array 등, 신속하면서도 민감도와 특이도가 높은 분자생물학적 방법을 이용하여 환자로부터 분리한 직접 검체에서 결핵균을 검출하는 방법의 이용이 점차 늘어나고 있는 추세이다. 또한 비결핵 항산균의 동정법으로 많이 사용되고 있는 방법 중의 하나가 PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA) 방법이다[Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000;38:2966-71]. PRA방법은 결핵균과 비결핵 항산균의 동정이 가능한 유전자 부위를 PCR로 증폭한 후, 적절한 제한효소를 처리하여 그 절편의 양상을 보고 균을 동정하는 방법이다. The most reliable diagnosis of tuberculosis is to prove infection by Mycobacterium tuberculosis, and therefore, the most reliable method is to detect MTB directly from clinical specimens. Therefore, the use of methods for detecting Mycobacterium tuberculosis in direct samples isolated from patients using molecular biology methods, such as PCR, real-time PCR, hybridization, and oligonucleotide arrays, has been increasing. In addition, one of the methods widely used for the identification of non-tuberculosis antibacterial bacteria is PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA) method [Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000; 38: 2966-71. The PRA method is a method for identifying bacteria by amplifying a gene region capable of identifying tuberculosis bacteria and non-tuberculosis antibacterial bacteria by PCR, and processing the appropriate restriction enzymes.
그러나 PRA방법은 결핵균과 비결핵 항산균 등 두 가지 이상의 균이 섞여 있는 경우에는 제한효소를 처리한 절편의 양상이 복합적으로 나타나기 때문에 동정이 불가능하다는 단점이 있다. 이러한 불편함을 보완하고자 최근에는 결핵균과 비결핵 항산균을 동시에 진단 및 동정할 수 있으며, 다양한 균이 섞여있는 경우에도 동정이 가능한 reverse blot hybridization assay (REBA)들이 개발되어 상용화되고 있다. REBA법은 검체 또는 균배양액으로부터 분리한 핵산의 특정 유전자 부위를 증폭한 후, 멤브레인에 고정화되어 있는 각각의 균종에 특이적인 oligonucleotide 프로브에 반응시켜 프로브와 PCR산물의 결합 여부를 검사함으로써 균을 동정하는 방법이다[Bunschoten A, Tiemersma E, Schouls L, Kampman E. Simultaneous Analyt. Biochem. 2000;285:156-62]. However, the PRA method has a disadvantage in that identification of fragments treated with restriction enzymes is impossible when two or more bacteria, such as tuberculosis bacteria and non-tuberculosis antibiotics, are mixed. In order to compensate for this inconvenience, in recent years, tuberculosis bacteria and non-tuberculosis anti-bacterial bacteria can be diagnosed and identified simultaneously, and reverse blot hybridization assays (REBA) have been developed and commercialized even when various bacteria are mixed. The REBA method identifies a bacterium by amplifying a specific gene region of a nucleic acid isolated from a sample or a culture medium, and then reacting with an oligonucleotide probe specific to each species immobilized on a membrane to test whether the probe and the PCR product are bound. Method [Bunschoten A, Tiemersma E, Schouls L, Kampman E. Simultaneous Analyt. Biochem. 2000; 285: 156-62.
관련 특허로 대한민국 특허공개번호 제1019990086567호는 '결핵균 및 비결핵균항산성균의 탐지용진단시약'에 관한 것으로, RNA 중합효소 β 서브유닛을 코딩하는 유전자(rpoB)를 표적으로 하여 결핵균군(Mycobacterium tuberculosis complex)과 비결핵 항산성균(Mycobacteria other than Tubercle bacilli: MOTT)을 감별-탐지할 수 있는 이중 중합효소 연쇄반응(duplex-polymerase chain reaction: duplex-PCR)용 프라이머쌍, 그를 이용한 결핵균군과 MOTT의 감별-탐지방법 및 전기 감별-탐지 방법으로서 결핵과 MOTT 감염여부를 진단하는 방법에 관한 것이며, 표적 DNA로서 rpoB 유전자를 이용하여 간편하고 효과적으로 결핵균군과 MOTT를 감별하여 탐지할 수 있는 이중 PCR용 프라이머 쌍을 개발하였다. 전기 두 특이 프라이머 쌍을 이용하여 동시에 PCR을 수행함으로써, 검체에 존재하는 주형 DNA의 종류에 따라 결핵균군 또는, MOTT의 rpoB DNA가 각기 다른 크기의 단일밴드로 증폭되고 이에 의하여, 그 크기에 따라 감염여부를 확인할 수 있어서, 프라이머쌍을 이용한 이중 PCR 방법에 의하면 모든 마이코박테리아에 단일 복제수(single copy)로 존재하면서, 매우 잘 보존되어 있는 rpoB 유전자 하나만을 표적으로 하여, 종래의 어떠한 방법보다도 간편하고 특이적으로 결핵균군과 MOTT를 감별-탐지할 수 있다고 기재되어 있다.
As a related patent, Korean Patent Publication No. 1019990086567 relates to 'Diagnostic Reagent for the Detection of Mycobacterium Tuberculosis and Non-tuberculosis Antibacterial Acid Bacteria,' targeting a gene (rpoB) encoding RNA polymerase β subunit (Mycobacterium tuberculosis complex). ) And a pair of primers for the duplex-polymerase chain reaction (duplex-PCR) that can discriminate and detect Mycobacteria other than Tubercle bacilli (MOTT), As a detection method and an electrical discrimination-detection method, the present invention relates to a method for diagnosing tuberculosis and MOTT infection, and developed a dual PCR primer pair that can easily and effectively detect tuberculosis bacteria and MOTT using rpoB gene as target DNA. It was. By simultaneously performing PCR using the two specific primer pairs, tubercle bacillus or MOTT rpoB DNA is amplified into single bands of different sizes according to the type of template DNA present in the sample, thereby infecting them according to their size. According to the dual PCR method using primer pairs, it is simpler than any conventional method by targeting only one rpoB gene that is very well conserved while being present as a single copy in all mycobacteria. It is specifically described that it is possible to differentially detect tuberculosis bacteria and MOTT.
본 발명은 상기의 문제점을 해결하고, 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 결핵균 및 비결핵 항산균의 구별방법을 제공하는 것이다.The present invention solves the above problems, and the object of the present invention is to provide a method for distinguishing tuberculosis bacteria and non-tuberculosis antibacterial bacteria.
본 발명의 다른 목적은 결핵균 및 비결핵 항산균의 구별용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for distinguishing Mycobacterium tuberculosis and non-tuberculosis antibacterial bacteria.
본 발명의 또 다른 목적은 결핵균 및 비결핵 항산균 구별용 키트를 제공하는 것이다Still another object of the present invention is to provide a kit for distinguishing Mycobacterium tuberculosis and non-tuberculosis antibacterial bacteria.
상기의 목적을 달성하기 위하여 본 발명은 a)검체 시료로부터 DNA를 분리하는 단계;b)프라이머로서 서열번호 1 및 서열번호 2를 사용하여, 상기 DNA로부터 rpoB 유전자 상의 유전자 단편을 PCR 증폭하는 단계;및c)서열번호 5 내지 29의 올리고머 프로브가 부착되어 있는 고체 서포트와 상기 단계 b)에서 얻어진 PCR 증폭산물을 하이브리드형성시키는 PCR-리버스 블랏 하이브리드형성 단계;를 포함하는 것을 특징으로 하는 결핵균 및 비결핵 항산균의 구별 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of: a) separating DNA from a sample; b) PCR amplifying a gene fragment on the rpoB gene from the DNA using SEQ ID NO: 1 and SEQ ID NO: 2 as a primer; And c) a PCR-reverse blot hybridization step of hybridizing the solid support to which the oligomeric probes of SEQ ID NOs: 5 to 29 are attached and the PCR amplification product obtained in step b). It provides a method for distinguishing antibacterial bacteria.
본 발명의 일 구현예에 있어서, 상기 방법은 서열번호 3 내지 4의 프라이머를 이용하여 PCR을 수행하는 단계를 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the method preferably further comprises performing a PCR using the primers of SEQ ID NOs: 3 to 4, but is not limited thereto.
본 발명의 다른 구현예에 있어서, 상기 비결핵 항산균은 상기 비결핵 항산균은 마이코박테리움 아비움(Mycobacterium. avium), Mycobacterium. intracellurare, Mycobacterium. scrofulaceum, Mycobacterium.abscessus, Mycobacterium.massiliense, Mycobacterium.chelonae, Mycobacterium.fortuitum/peregrinum/ulceranse/marinum, Mycobacterium.ulcerans/marinum, Mycobacterium.kansasii, Mycobacterium.genevans/simiae, Mycobacterium.terrae, Mycobacterium.nonchromogenicum, Mycobacterium.celatum, Mycobacterium.gordonae, Mycobacterium. szulgai, Mycobacterium.mucogenicum, Mycobacterium.aubagnens 및 Mycobacterium.bolletii로 구성된 군으로부터 선택된 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the non-TB tuberculosis is the non-TB tuberculosis mycobacterium avium (Mycobacterium. Avium), Mycobacterium. intracellurare, Mycobacterium. scrofulaceum, Mycobacterium.abscessus, Mycobacterium.massiliense, Mycobacterium.chelonae, Mycobacterium.fortuitum / peregrinum / ulceranse / marinum, Mycobacterium.ulcerans / marinum, Mycobacterium.kansasii, Mycobacterium.genevans, Mycobacterium.genevans, Mycobacterium. celatum, Mycobacterium.gordonae, Mycobacterium. preferably selected from the group consisting of szulgai, Mycobacterium.mucogenicum, Mycobacterium.aubagnens and Mycobacterium.bolletii.
본 발명의 또 다른 구현예에 있어서, 상기 방법은 M. abscessus와 M. massiliense 구별에 이용될 수 있는 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the method may be used to distinguish between M. abscessus and M. massiliense , but is not limited thereto.
또 본 발명은 서열번호 4 내지 서열번호 29에 기재된 서열을 갖는 결핵균 및 비결핵 항산균 구별용 올리고머 프로브 조성물을 제공한다.In another aspect, the present invention provides an oligomeric probe composition for distinguishing Mycobacterium tuberculosis and non-tuberculosis antibacterial bacterium having the sequence of SEQ ID NO: 4 to SEQ ID NO: 29.
또 본 발명은 상기 본 발명의 조성물이 부착된 고체 서포트를 제공하며, 일 구현예에 있어서, 상기 고체 서포트는 막, 슬라이드, 또는 웰 플레이트인 것이 바람직하나 이에 한정되지 아니한다.In another aspect, the present invention provides a solid support to which the composition of the present invention is attached, and in one embodiment, the solid support is preferably a membrane, a slide, or a well plate, but is not limited thereto.
또 본 발명은 서열번호 1 내지 2에 기재된 프라이머, 및 제 4항에 기재된 올리고머 프로브 조성물을 유효성분으로 포함하는 결핵균 및 비결핵 항산균 검출용 키트를 제공한다.The present invention also provides a kit for detecting Mycobacterium tuberculosis and non-tuberculosis antibacterial bacterium comprising the primers of SEQ ID NOS: 1 and 2, and the oligomeric probe composition of
상기 키트는 PCR 증폭 반응을 수행하기 위한 시약으로, DNA 폴리머라제, dNTPs 및 버퍼를 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.The kit is a reagent for performing a PCR amplification reaction, and preferably further includes DNA polymerase, dNTPs, and a buffer, but is not limited thereto.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명에서는 신속하고 정확하며 결핵 및 비결핵 항산균을 검출하고 동정할 수 있는 REBA Myco-ID의 임상적 유용성 평가 및 검출율을 비교하고자 하였다.In the present invention, it was intended to compare the clinical usefulness evaluation and detection rate of REBA Myco-ID that can detect and identify tuberculosis and non-tuberculosis antibacterial bacteria quickly and accurately.
이를 위하여 148개의 항산성 염색 양성 호흡기 검체를 대상을 하여 역교잡의 원리를 기초로 한 본 발명의 REBA Myco-ID 를 수행하고 그 결과 동일한 직접 검체를 배양한 배양액으로 결핵균과 비결핵균을 구별할 수 있는 키트인 MTB-ID의 결과와 균종 동정 키트인 PRA Myco-ID의 결과와 비교하였다.For this purpose, the REBA Myco-ID of the present invention based on the principle of reverse hybridization in 148 acid-staining positive respiratory specimens. The results were compared with the results of MTB-ID, a kit for distinguishing Mycobacterium tuberculosis and non-tuberculosis, from the culture of the same direct sample and PRA Myco-ID.
그 결과, 항산성염색 양성에서 판독된 59검체의 결핵균과 89검체의 비결핵 항산균의 REBA Myco-ID 의 민감도는 100%로 나왔으나 MTB/NTM PCR에서 비결핵 항산균으로 나왔던 하나의 배양균주가 REBA Myco-ID 에서는 결핵균을 포함한 M. szulgai M. gordonae 등이 섞여 있는 것으로 검출되어 REBA Myco-ID는 직접 검체 내에 두 개 이상의 균이 있는 경우에도 모든 균종을 다 검출 및 동정할 수 있는 유용한 키트임을 보여주었다. 또한, NTM의 검출분포를 확인해 본 결과 M. intracellulare 28검체(32.2%), M. avium 21검체(24.1%), M. massiliense 19검체(21.8%), M. abscessus 15검체(17.2%), M. kansasii 1검체(1.2%)순으로 검출되었으며, M. avium 와 M. mucogenicum, M. avium 와 M. abscessus , M. intracellulare 와 M. scrofulaceum이 각각 1검체(1.2%)로 2개 이상의 균종이 혼합되어 검출되는 경우도 확인이 되었다.As a result, the sensitivity of REBA Myco-ID of the tuberculosis bacteria of 59 samples and the non-tuberculosis mycobacterium of 89 samples, which were positive in anti-acid staining, was 100%, but one culture strain that emerged as non-tuberculous acid bacteria in MTB / NTM PCR. REBA Myco-ID is detected as a mixture of M. szulgai M. gordonae , including tuberculosis bacteria, and REBA Myco-ID is a useful kit that can detect and identify all species even if there are two or more bacteria in the sample. Showed that. In addition, the detection distribution of NTM revealed that 28 samples of M. intracellulare (32.2%), 21 samples of M. avium (24.1%), 19 samples of M. massiliense (21.8%), 15 samples of M. abscessus (17.2%), M. kansasii 1 samples (1.2%) were detected, and M. avium and M. mucogenicum, M. avium and M. abscessus , M. intracellulare and M. scrofulaceum were 1 specimen (1.2%), respectively. It was also confirmed that the mixture was detected.
따라서 본 발명의 REBA Myco-ID은 전통적인 방법보다 빠르게 정확하게 결핵균과 비결핵 항산균의 감별 및 동정이 가능하며 검체 내에 한 종류의 균이 존재하는 경우는 물론이고 결핵균과 비결핵 항산균이 섞여 있는 경우나 여러 종의 비결핵 항산균이 섞여있는 경우, 동시에 각각의 균종의 검출 및 동정이 가능함을 확인하였다. 따라서 REBA Myco-ID는 원인균을 정확하게 진단함으로써 환자의 적절한 치료에 유용한 정보를 제공할 수 있을 것으로 판단된다.Therefore, REBA Myco-ID of the present invention is able to discriminate and identify tuberculosis bacteria and non-tuberculosis antibacterial bacteria more accurately than the conventional method, and when tuberculosis bacteria and non-tuberculosis antibacterial bacteria are mixed as well as the presence of one type of bacteria in the sample. B. When several non-tuberculosis mycobacteria were mixed, it was confirmed that the detection and identification of each species was possible at the same time. Therefore, the REBA Myco-ID is expected to provide useful information for proper treatment of patients by accurately diagnosing the causative organism.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 REBA Myco-ID 키트는 종특이 다형성이 존재하는 rpoB 유전자 부위를 amino group이 부착된 프라이머로 증폭하고 얻어진 PCR산물을 종특이 프로브가 부착되어있는 멤브레인과 반응시켜 결핵균을 포함한 20종의 주요 비결핵 항산균(M. avium , M. intracellulare , M. scrofulaceum , M. abscessus , M. massiliense, M. chelonae , M. fortuitum complex , M. ulcerans /M. marinum , M. kansasii , M. genavense/M. simiae , M. terrae /M. nonchromogenicum , M. celatum , M. gordonae , M. szulgai , M. mucogenicum, M. aubagnense 및 Mycobacterium.bolletii)을 검출 및 동정할 수 있는 키트이다.REBA Myco-ID Kit of the invention are 20 species to species react with the membrane in the PCR product amplifying the rpo B gene region with the amino group attached to the primer and obtained by the specific polymorphisms present is attached to the species-specific probes, including M. tuberculosis Major non-TB tuberculosis bacteria ( M. avium , M. intracellulare , M. scrofulaceum , M. abscessus , M. massiliense, M. chelonae , M. fortuitum complex , M. ulcerans / M. marinum , M. kansasii , M. genavense / M. simiae , M. terrae / M. nonchromogenicum , M. celatum , M. gordonae , M. szulgai , M. mucogenicum, M. aubagnense And Mycobacterium.bolletii).
본 발명에서는 개발된 인 REBA Myco-ID를 이용하여 임상에서 분리된 직접 검체에서 1) 결핵균의 검출 및 비결핵 항산균과의 감별을 시도하고 2) 검체에서 검출되는 비결핵 항산균의 빈도 및 분포에 대한 자료를 확보하며 3) 검체에 존재하는 결핵균과 비결핵 항산균의 mixed infection에 대한 자료 확보 및 4) 배양과정에서의 검체의 오염률을 파악하고자 하였다. In the present invention, 1) detection of Mycobacterium tuberculosis and differentiation from non-tuberculosis mycobacterium in direct samples isolated from clinical trials using the developed phosphorus REBA Myco-ID, and 2) frequency and distribution of non-tuberculosis mycobacterium detected in the sample Data were collected and 3) data were collected on the mixed infection of Mycobacterium tuberculosis and non-tuberculosis acid bacillus, and 4) the contamination rate of the sample in the culture process was investigated.
결핵환자의 발생은 주로 활동성 결핵을 가진 환자의 기침이나 재채기 등으로 인해 공기 중으로 나온 비말핵에 의해 전파되므로 활동성 결핵환자를 조기에 진단하여 치료하는 것이 결핵의 전염을 예방하는데 중요하다고 알려져 있다[Gaby E. pfyffer. Mycobacterium: General Characteristics, Laboratory Detection, and Staining procedures. In: Patrick R. murray, ed, manual of Clinical Microbiology. 9th ed, Washington, DC; American Society for Microbiology, 2007:543-72]. 국내의 경우 결핵의 유병률이 높고 비결핵 항산균 질환의 빈도가 낮아 항산균 도말양성일 경우 대부분 결핵으로 간주하고 항결핵제 치료를 시행하는 것이 일반적이었으나, 최근 국내에서도 비결핵 항산균의 분리율 및 비결핵 항산균 질환이 증가함에[Park CM, Heo SR, Park KU, Song JH, Lee JH, Lee CT, et al. Isolation of Nontuberculosis mycobacteria using polymerase chain reaction-restriction fragment length polymorphism. Korean J Lab Med 2006;26:161-7;Koh WJ, Kwon OJ, Ham HS, Suh GY, Chung MP, Kim HJ, et al. Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens. Korean J Med 2003;65:10-21] 따라서 비결핵 항산균을 구별 및 동정할 수 있는 진단법의 필요성이 커지고 있다.The incidence of tuberculosis patients is mainly transmitted by airborne droplet nuclei due to coughing or sneezing in patients with active tuberculosis. Therefore, early diagnosis and treatment of active tuberculosis patients are known to be important in preventing the spread of tuberculosis [Gaby E. pfyffer. Mycobacterium: General Characteristics, Laboratory Detection, and Staining procedures. In: Patrick R. murray, ed, manual of Clinical Microbiology. 9 th ed, Washington, DC; American Society for Microbiology, 2007: 543-72. In Korea, due to high prevalence of tuberculosis and low incidence of non-tuberculosis mycobacterial diseases, most cases of antibacterial bacterium smear were considered tuberculosis and treated with anti-tuberculosis drugs. With increasing disease [Park CM, Heo SR, Park KU, Song JH, Lee JH, Lee CT, et al. Isolation of Nontuberculosis mycobacteria using polymerase chain reaction-restriction fragment length polymorphism. Korean J Lab Med 2006; 26: 161-7; Koh WJ, Kwon OJ, Ham HS, Suh GY, Chung MP, Kim HJ, et al. Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens. Korean J Med 2003; 65: 10-21] Therefore, there is a growing need for diagnostic methods to identify and identify non-tuberculosis mycobacteria.
최근에는 결핵을 진단하는 데 있어서 PCR 등을 이용한 분자생물학적 검사법들이 많이 사용되고 있다. 또한, 결핵 진단을 위한 직접 검체로는 객담을 가장 많이 사용하는데, 객담 자체는 일반적인 PCR검사의 민감도가 낮으며 대부분의 PCR법은 결핵균이나 비결핵 항산균만 구별이 가능하며, 비결핵 항산균을 동정하기 위해서는 PRA방법처럼 추가적인 실험이 필요하다. 하지만 PRA방법은 결핵균과 비결핵 항산균이 섞여있거나 서로 다른 비결핵 항산균들이 섞여 있는 경우는 동정이 힘들다는 단점이 있다. Recently, molecular biology tests using PCR are widely used to diagnose tuberculosis. In addition, sputum is most commonly used as a direct specimen for the diagnosis of tuberculosis, and sputum itself has low sensitivity of general PCR tests, and most PCR methods can distinguish only tuberculosis bacteria and non-tuberculosis antibiotics. To identify, additional experimentation is required, such as the PRA method. However, the PRA method has a disadvantage in that it is difficult to identify when tuberculosis bacteria and non-tuberculosis antibiotics are mixed or different non-tuberculosis antibiotics are mixed.
본 발명의 REBA Myco-ID는 이러한 문제점을 해결하고자 검체에서 biotin이 표지된 primer로 PCR을 한 후 그 산물을 가지고 5'아민기가 있는 본 발명의 프로브가 부착된 strip에 붙여 결핵균과 비결핵항산균의 검출 및 비결핵 항산균의 감별까지 가능하도록 개발되었고 본 발명에서 직접 검체를 대상으로 하여 키트의 임상적 유용성을 평가해보았다.REBA Myco-ID of the present invention is to solve this problem by PCR with a biotin-labeled primer in the sample and then with the product attached to the strip attached to the probe of the present invention having a 5 'amine group Mycobacterium tuberculosis and non-TB tuberculosis It was developed to be able to detect and discriminate non-tuberculosis mycobacterium tuberculosis, and the clinical usefulness of the kit was evaluated directly in the present invention.
서울삼성병원에서 AFB smear와 배양검사 등이 끝난 148 예의 직접 검체와 동일한 검체의 배양액을 제공받았다. 총 148검체 중 NTM이 분리된 검체는 89검체(60%)로 이는 국내 NTM 분리율이 2000년 이후 26~46% 정도라는 최근의 보고[Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009;67:199-204]와 비교하였을 때 다소 높은 비율이나 이는 검체를 제공한 병원이 다른 병원에 비해서 NTM의 환자들이 비교적 많다는 기존의 보고와 일치하는 결과이다[Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2008;74:120-31]. 제공된 검체로 MTB과 NTM을 구분하는 PCR 키트인 MTB-ID (M&D, Korea)를 수행하고 NTM으로 판정된 검체를 대상으로 하여 NTM 균 종 동정이 가능한 REBA Myco-ID와 PRA Myco-ID (M&D, Korea)를 수행하여 그 결과를 비교하였다. Seoul Samsung Hospital received the same culture medium as 148 cases of AFB smear and culture test. Of the 148 samples, NTM was isolated from 89 samples (60%), which is a recent report that domestic NTM separation rate was 26 ~ 46% since 2000 [Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009; 67: 199-204], although the ratio is somewhat higher, which is consistent with previous reports that the hospital that provided the sample had relatively more patients with NTM than other hospitals [Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2 008; 74: 120-31. Perform MTB-ID (M & D, Korea), a PCR kit that distinguishes MTB and NTM with the provided sample, and targets the REBA Myco-ID and PRA Myco-ID (M & D, Korea) to compare the results.
본 발명에서 결핵균과 비결핵 항산균 MTB-ID의 결과와 PRA Myco-ID의 결과, 그리고 REBA Myco-ID의 결과가 두 검체를 제외한 모든 검체에서 일치하였다.In the present invention, the results of M. tuberculosis and non-TB tuberculosis MTB-ID, PRA Myco-ID, and REBA Myco-ID were consistent in all samples except the two samples.
MTB-ID에서 비결핵 항산균으로 판정된 한 검체(도말 결과상 1+)의 REBA Myco-ID 결과가 결핵균을 포함한 M. szulgai , M. gordonae , M. genavense /M. simmiae 등 비결핵 항산균이 섞여 나와(도 2A) 서로 불일치한 결과를 보였는데, 이 경우 PRA상에는 복합적인 밴드 양상을 보여 Un-ID로 판정되었고(도 2B와 표 2.), sequencing결과에서도 여러 균종의 염기서열이 섞여 있어서 판정이 불가능하였다. 이 검체의 환자의 정보를 확인해 본 결과 과거에 결핵균에 의한 결핵으로 진단되어 이미 치료한 상태였다. 따라서 객담으로부터 바로 검사를 한 REBA Myco-ID 에서는 결핵균 뿐만 아니라 비결핵 항산균을 모두 검출할 수 있었으나 배양하는 과정에서 비결핵 항산균의 배양에 의해 결핵균의 배양이 가려진 것으로 판단된다(표 2). 또한 다른 1개의 검체는 MTB-ID에서는 비결핵 항산균으로, REBA Myco-ID로는 아무 것도 나오지 않았는데(도 2C), 액체배지를 이용하여 PRA를 한 결과 N. farcinia로 검출된 것을 확인 할 수 있었다(도 2D). 이는 MTB-ID에서 타켓으로 하고 있는 유전자 중의 하나인 rpoB 유전자에서 Mycobacteria의 공통서열이 위치하고 있는 부위가 Nocardia spp., Rhodococcus spp. 등과 유사한 염기서열을 가지고 있기 때문에 PCR 결과상 Nocardia도 비결핵 항산균으로 검출되나, REBA Myco-ID는 동일한 rpoB 유전자 부위를 사용하기는 하지만 특이적인 프로브를 이용해 검출하기 때문에 더욱더 민감하고 특이적으로 mycobacteria를 우선적으로 동정하므로, PRA에서 Nocardia spp. 로 동정된 검체는 REBA Myco-ID에서 아무런 시그널도 보이지 않은 것으로 판단된다.REBA Myco-ID results from one specimen (1+ in the smear) determined as non-tuberculosis mycobacterium in MTB-ID showed M. szulgai , M. gordonae , M. genavense / M. Non-tuberculosis acidophilus such as simmiae were mixed (Fig. 2A) and showed inconsistent results. In this case, PRA showed a complex band pattern and was determined to be Un-ID (Fig. 2B and Table 2.) Judgment was not possible due to the mixed nucleotide sequence of the species. As a result of checking the patient's information, the patient was diagnosed with tuberculosis in the past and already treated. Therefore, the REBA Myco-ID tested directly from sputum was able to detect not only tuberculosis bacteria but also non-tuberculosis mycobacterium. In addition, the other sample was non-tuberculosis antibacterial bacterium in MTB-ID, and none appeared as REBA Myco-ID (FIG. 2C). As a result of PRA using liquid medium, N. farcinia was detected. (FIG. 2D). Nocardia is the site where the common sequence of Mycobacteria is located in rpo B gene, one of the target genes of MTB-ID. spp ., Rhodococcus spp . As a more sensitive and specific but, because PCR results the Nocardia they have a similar nucleotide sequence is also detected in the non-tuberculosis mycobacteria, because REBA Myco-ID is to use the same rpo B gene regions but to detect using the specific probe Nocardia in PRA, because it preferentially identifies mycobacteria spp. The samples identified as did not show any signal from REBA Myco-ID.
또한 본 발명의 결과에는 보여주지 않았지만 대조군으로 사용하기 위하여 AFB smear와 배양검사 모두에서 음성으로 나온 정상인 25검체를 대상으로 REBA Myco-ID 에 대한 특이도 실험을 진행하였으며 병원의 결과와 동일하게 모두 음성으로 나와 REBA Myco-ID가 우수한 특이도를 가지고 있음을 확인할 수 있었다.In addition, although not shown in the results of the present invention, the specificity test for REBA Myco-ID was performed on 25 normal subjects that were negative in both AFB smear and culture test for use as a control group. It was confirmed that REBA Myco-ID has excellent specificity.
한편 본 발명에서 비결핵 항산균의 동정분포를 확인해 보면 배양 검체에서 M. intracellulare가 28검체 (32.2%)로 가장 높게 검출되었으며, M. avium 이 21검체 (24.1%)와 M. massiliense 19검체 (21.8%) 그리고 M. abscessus 15검체 (17.2%)순으로 검출되었는데, 이는 미국과 일본에서 비결핵 항산균 폐질환의 가장 흔한 원인균인 M. avium - intracellulare complex (MAC) 가 전체 비결핵 항산균 폐질환의 60~80%를 차지하며[Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009;67:199-204;Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2008;74:120-31], 우리나라에서도 비결핵 항산균 폐질환 원인균의 50~60%를 차지하고 있다는 보고[Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009;67:199-204;Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2008;74:120-31;Lee HW, Kim MN, Shim TS, Bae GH, Pai CH. Nontuberculous mycobacterial pulmonary infection in immunocompetent patients. Tuberc Respir Dis 2002;53:173-82;Koh WJ, kwon OJ, Lee KS. Nontuberculous mycobacterial pulmonary diseases in immunocompetent patients. Korean J Radiol 2002;2:145-5725-28]와 본 실험에서의 49검체(56.3%)의 결과가 유사하게 나타났다. 또한 M. massiliense와 M. abscessus를 포함한 M. abscessus complex의 분포비율도 34 검체 (39%)로 높게 나타났는데 이는 M. abscessus complex의 분포비율이 M. avium - intracellulare complex의 분포비율과 거의 동등한 수로 나타나며 M. abscessus를 포함한 M. massiliense의 중요성이 점점 증가된다는 보고[Koh WJ, Jeon K, Lee NY, Kim BJ, Kook YH, Lee SH, et al. Clinical Significance of Differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am J Respir Crit Care med. 2010;183:405-10]와 유사한 결과를 나타낸 것을 확인할 수 있었으며 REBA Myco-ID는 MAC뿐만 아니라 PRA Myco-ID에서도 구별이 안되는 M. abscessus를 포함한 M. massiliense의 분리검출이 가능하여 그 임상적 유용성이 매우 높음을 확인할 수 있었다(표 3).On the other hand, the identification distribution of non-tuberculosis mycobacterium in the present invention, M. intracellulare was detected as the highest in 28 (32.2%) of cultured samples, and M. avium 21 specimens (24.1%) and M. massiliense Nineteen specimens (21.8%) and M. abscessus 15 samples (17.2%) were detected in the US and Japan, where M. avium - intracellulare complex (MAC), the most common cause of non-TB bacteriostatic lung disease, was found in the US. It accounts for 60 ~ 80% of the acidic lung disease [Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009; 67: 199-204; Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2 008; 74: 120-31], reported in Korea that it accounts for 50-60% of the causative agents of non-tuberculosis mycobacterial lung disease [Lee JK, Kwon HY, Kwon JK, Lee HJ, Lee DW, Lee YJ, et al. Recovery Rate of Nontuberculous Mycobacteria and the Clinical Course of Nontuberculous Mycobacterial Pulmonary Disease at a Secondary Hospital. Tuberc Respir Dis. 2009; 67: 199-204; Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med . 2 008; 74: 120-31; Lee HW, Kim MN, Shim TS, Bae GH, Pai CH. Nontuberculous mycobacterial pulmonary infection in immunocompetent patients. Tuberc Respir Dis 2002; 53: 173-82; Koh WJ, kwon OJ, Lee KS. Nontuberculous mycobacterial pulmonary diseases in immunocompetent patients. Korean J Radiol 2002; 2: 145-5725-28] and 49 specimens (56.3%) were similar in this experiment. In addition, the distribution ratio of M. abscessus complex including M. massiliense and M. abscessus was 34 samples (39%), indicating that the distribution ratio of M. abscessus complex was almost equal to that of M. avium - intracellulare complex. And increased importance of M. massiliense including M. abscessus [Koh WJ, Jeon K, Lee NY, Kim BJ, Kook YH, Lee SH, et al. Clinical Significance of Differentiation of Mycobacterium massiliense from Mycobacterium abscessus . Am J Respir Crit Care med. 2010; 183: 405-10] and showed similar results, and REBA Myco-ID is not only MAC M. massiliense including M. abscessus , which is indistinguishable from PRA Myco-ID, can be isolated and detected, and its clinical usefulness is very high (Table 3).
또한 본 발명은 선행 특허에 비하여 REBA Myco-ID 에서 M. massiliense의 구별이 가능하다는 것이다 M. abscessus는 일반적인 항결핵제뿐만 아니라 많은 항생제에 내성을 보이며 약제감수성검사에서 clarithromycin, amikacin, cefoxitin 등에 대해서만 감수성을 보이는데, 최근 M. abscessus는 M. abscessus , M. massiliense 그리고 M. bolletii라는 3가지 다른 균으로 이루어져 있다는 것이 밝혀졌습니다. 각 균종의 분포는 나라에 따라 다르며, 국내에서는 M. abscessus와 M. massiliense이 각각 50% 내외를 차지하며, M. bolletii는 매우 드물다.In addition, the present invention compared to the prior patent REBA Myco-ID M. in that it is possible to distinguish the massiliense M. abscessus is common line drugs but also to many antibiotics showed a resistance exhibit sensitivity only the like clarithromycin, amikacin, cefoxitin in the drug sensitivity test, recently M. abscessus is M. abscessus, M. It turns out that it consists of three different bacteria, massiliense and M. bolletii. M. abscessus and M. massiliense account for about 50% of each species, and M. bolletii is very rare in Korea.
M. abscessus는 clarithromycin에 노출되면 erm 유전자가 발현되어 clarithromycin에 대한 유도내성이 발현된다.따라서 약제감수성검사에서 clarithromycin 감수성을 보이더라도 실제 환자에게 투여하면 효과가 소실될 수 있다.이와 달리 M. massiliense는 이러한 clarithromycin 유도내성이 발견되지 않아 항생제 치료 성적이 M. abscessus에 비해 매우 높다고 보고되고 있다. 따라서 M. abscessus와 M. massiliense의 구별은 중요하다고 보여진다. M. abscessus when exposed to clarithromycin is erm gene expression is induced resistance to clarithromycin is developed, so even if it seems to clarithromycin sensitivity in drug susceptibility test the effect may be lost when the actual administration to the patient. In contrast, the M. massiliense Since clarithromycin-induced resistance was not found, antibiotic treatment results were reported to be much higher than that of M. abscessus . Therefore, the distinction between M. abscessus and M. massiliense seems to be important.
또한 선행 특허의 Dual-ID는 MTB/NTM의 구별외에 Rifampicin 약제내성의 여부까지도 알 수 있다는 장점이 있는 내용이나 PCR size가 530bp로 커서 배양에서는 그 결과를 잘 알 수 있으나 (도 3) 객담 등의 직접검체에서는 결과가 잘 나오지 않아 (도 4-5) MTB와 NTM만을 구별할 수 있는 REBA Myco-ID를 PCR size를 줄여서(270bp) 배양뿐만 아니라 직접검체에서도 구별할 수 있도록 고안되었다(도 6-7) In addition, the Dual-ID of the prior patent has the advantage of knowing whether Rifampicin drug resistance is not only distinguished from MTB / NTM, but the PCR size is 530bp, so the result can be well understood in culture (FIG. 3). The results were not well obtained in the direct sample (Fig. 4-5) by reducing the PCR size (270bp) of the REBA Myco-ID that can distinguish only MTB and NTM was designed to be distinguished from the direct sample as well (Fig. 6- 7)
본 발명을 통해서 알 수 있는 바와 같이, 본 발명의 REBA Myco-ID는 객담 등의 직접 검체에서 결핵균 및 비결핵 항산균의 구별이 가능하고, 비결핵 항산균의 동정까지 가능하기 때문에 빠른 시간 내에 원인균을 정확히 동정함으로써 비결핵 항산균에 작용할 수 있는 올바른 약제를 처방할 수 있도록 유용한 정보를 제공하며, 본 발명에서 보았듯이 액체배양의 사용이 점차 늘어남에 따라 결핵균과 빨리 자라는 비결핵 항산균과 섞여있는 경우 비결핵 항산균으로 오진되는 것을 방지할 수 있을 것으로 판단된다. 따라서 REBA Myco-ID는 결핵균 및 비결핵 항산균 감염증의 진단에 매우 유용하게 사용될 수 있을 것으로 판단된다.As can be seen through the present invention, REBA Myco-ID of the present invention can distinguish between tuberculosis bacteria and non-tuberculosis mycobacteria in direct samples such as sputum, and it is possible to identify non-tuberculosis mycobacterium bacteria, so it is possible to identify causative bacteria in a short time. It provides accurate information to correctly identify the correct drug that can act on non-tuberculosis mycobacterium by accurately identifying and, as seen in the present invention, as the use of liquid culture increases, it is mixed with mycobacterium tuberculosis and non-tuberculosis mycobacterium that grows quickly. In this case, it can be prevented from being misdiagnosed as non-tuberculosis antibacterial bacteria. Therefore, REBA Myco-ID can be very useful for the diagnosis of M. tuberculosis and non-TB tuberculosis.
도 1은 AFB smear에 의한 임상 객담 샘플의 REBA Myco-ID 결과를 나타낸 그림.
도 2는 NTM 동정 테스트의 비교 그림. (A) 객담 DNA를 사용한 REBA Myco-ID. lane 1, M. szulgai , M. gordonae, 및 MTB 소량 혼합. (B) 배양 DNA를 가지는 M&D PRA를 가지는 Myco-ID PCR-RFLP 분석(PRA). 레인 I에서, Msp I를 사용한 150-80의 밴드 패턴을 가지는 Un-ID, 혼합된 밴드 패턴이 배양에서 혼합된 NTM군의 존재를 시사하는 것으로 관찰될 수 있음.따라서 그 NTM 종은 결론적으로 동정될 수 없음. M, PRA size marker (M&D, Wonju, Korea), lane 1, Un-ID. (C) 객담 DNA를 사용한 REBA Myco-ID. lane 1, Un-ID. (D) 배양된 DNA를 가지는 M&D PRA Myco-ID를 사용한 PCR-RFLP analysis (PRA). lane 1, Msp I를 사용한 160-130-42의 밴드 패턴을 가지는 N. farcinia . lane 2, Hae III를 사용한 130-60의 밴드 패턴을 가지는 N.farcinia.. M, PRA 사이즈 마커(M&D, Wonju, Korea).
도 3은 선행 특허의 배양 결과를 나타낸 그림.
도 4-5는 선행특허의 방법을 이용한 결과, 객담 등의 직접검체에서는 결과가 잘 나오지는 않는다는 것을 보여주는 그림.
도 6 및 7은 MTB와 NTM만을 구별할 수 있는 REBA Myco-ID를 PCR size를 줄여서(270bp)배양뿐만 아니라 직접검체에서도 구별할 수 있다는 것을 보여주는 그림.
도 8은 본 발명의 프로브와 프라이머를 이용한 객담 결과를 나타낸 그림. 1 is a diagram showing the results of REBA Myco-ID of clinical sputum samples by AFB smear.
2 is a comparative illustration of the NTM identification test. (A) REBA Myco-ID using sputum DNA.
Figure 3 shows the culture results of the prior patent.
Figure 4-5 is a result showing that the results of using the method of the prior patent, the direct sample such as sputum does not come out well.
6 and 7 show that REBA Myco-ID, which can distinguish only MTB and NTM, can be distinguished from a direct sample as well as culture by reducing PCR size (270bp).
Figure 8 is a diagram showing the sputum results using the probe and primer of the present invention.
이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.
본 발명에 사용된 프라이머와 프로브는 바이오니아(대전, 대한민국)로부터 합성이 되었으며 제넷바이오(논산, 대한민국)로부터 제조된 2X PCR Taq DNA polymerase(Prime Taq DNA polymerase 1unit/10, 2X reaction buffer, 4mM MgCl2, enzyme stabilizer, sediment, loading dye, pH9.0, 0.5mM of dATP, dCTP, dGTP, dTTP)를 사용하여 PCR 증폭을 진행하였다. Primers and probes used in the present invention were synthesized from Bioneer (Daejeon, Korea) and prepared from Genenet Bio (Nonsan, Korea) 2X PCR Taq DNA polymerase (Prime Taq DNA polymerase 1unit / 10, 2X reaction buffer, 4mM MgCl2, PCR amplification was performed using enzyme stabilizer, sediment, loading dye, pH9.0, 0.5mM of dATP, dCTP, dGTP, dTTP).
실시예Example 1. 검체 1. Sample
2009년 3월부터 4월까지 서울삼성병원에서 항산균 도말검사와 배양검사가 동시에 의뢰된 검체 중 항산성 염색결과가 trace 이상으로 판독된 기관지 세척액 13예와 객담 135예를 포함하여 총 148 임상검체를 대상으로 하였다.
A total of 148 clinical specimens including 13 bronchial lavage fluids and 135 sputum samples whose anti-acid staining results were traced out of the specimens requested for both antimicrobial smear and culture tests at Seoul Samsung Hospital from March to April 2009 Was targeted.
실시예Example 2. 검체처리 2. Sample processing
제조사가 제공하는 실험방법에 따라 검체로부터 핵산을 분리하였다. 호흡기 검체에 검체와 동량의 4% NaOH를 넣고 잘 혼합한 다음 30분간 실온 방치 후 3,000 rpm에서 30분간 원심분리하였다. 원심분리한 상층액은 버리고 남은 균체를 삼등분하여 각각 항산균 도말, 배양, 그리고 핵산분리에 사용하였다. 수집된 균체에 REBA Myco-ID 키트 내에 포함되어 있는 DNA extraction solution 100 ㎕를 넣은 후 1분간 vortex하고, 100℃에서 10분간 가열한 후, 실온에서 13,000 rpm으로 3분간 원심분리하여 얻은 상층액을 취하는 방법을 이용하여 핵산을 분리하였다. 분리된 핵산은 REBA Myco-ID를 수행하기 위한 PCR의 주형으로 사용되었다.Nucleic acid was isolated from the sample according to the experimental method provided by the manufacturer. 4% NaOH was added to the respiratory sample and the same amount was mixed well, and then allowed to stand at room temperature for 30 minutes, followed by centrifugation at 3,000 rpm for 30 minutes. The supernatant, which had been centrifuged, was discarded and the remaining cells were divided into three and used for smearing, culturing, and nucleic acid separation. 100 μl of the DNA extraction solution contained in the REBA Myco-ID kit was added to the collected cells and vortexed for 1 minute, heated at 100 ° C. for 10 minutes, and centrifuged at 13,000 rpm for 3 minutes at room temperature to obtain the supernatant obtained. Nucleic acid was isolated using the method. The isolated nucleic acid was used as a template for PCR to perform REBA Myco-ID.
실시예Example 3. 3. 항산균Antibiotic acid 도말 및 배양 Smear and culture
결핵균 검출을 위한 항산균 도말 검사와 배양검사는 서울삼성병원에서 수행되었다. 항산균 도말검사는 Carbol-fuchsin을 이용한 Ziehl-Neelsen 염색법을 이용하였고 그 결과는 미국 질병예방통제국의 기준에 따라 판독하였다. 배양검사는 NaOH 처리하여 수거한 균체를 BACTEC MGIT 960 (Becton Dickinson Microbiology System, Sparks, Md, USA)에 접종함으로써 검체 내에 포함된 원인균을 배양하였다.
Antimicrobial smear and culture tests were performed at Seoul Samsung Hospital. Antimicrobial smears were tested using Ziehl-Neelsen staining with Carbol-fuchsin and the results were read according to the US Centers for Disease Control and Prevention. The culture test was inoculated with BOHEC MGIT 960 (Becton Dickinson Microbiology System, Sparks, Md, USA) to collect the NaOH-treated cells to culture the causative bacteria contained in the sample.
실시예Example 4. 4. 항산균의Antibacterial 분리 및 동정 Isolation and identification
직접 검체를 BACTEC MGIT 960 system (Becton Dickinson Microbiology System, Sparks, Md, USA)에 접종하고 37℃ 에서 배양한 후, 배양액의 일부를 취하여 100℃ 에서 20분간 가열하고 13,000rpm에서 5분간 원심분리한 후 상층액을 취하는 방법을 이용하였다. MTB-ID (M&D, Wonju, Korea)를 이용한 분자진단방법으로 결핵균과 비결핵 항산균을 구분하였고, PRA Myco-ID (M&D, Wonju, Korea)를 이용하여 비결핵 항산균 균종을 동정하였다[Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000;38:2966-71]. PRA Myco-ID는 제조사가 제공하는 실험방법에 따라 수행하였으며, 실험방법은 다음과 같다. 추출한 핵산은 이중-중합효소연쇄반응을 시행한 후 360 bp의 증폭산물을 전기영동으로 확인하였다. 증폭산물을 Msp I 효소로 처리하여 4% 아가로오즈 겔에 전기영동한 후 나타나는 핵산 절편 양상을 제조사가 제공하는 마이코박테리아 및 노카디아 동정 알고리즘에 따라 분석하였다. Msp I 효소로 처리에 의한 핵산 절편 양상으로 정확한 균동정이 안되는 경우 Hae III, Alu I등의 추가적인 제한 효소로 처리하여 얻은 핵산 절편 양상으로 균동정을 확인하였다[Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000;38:2966-71].
Directly inoculate the BACTEC MGIT 960 system (Becton Dickinson Microbiology System, Sparks, Md, USA), incubate at 37 ° C, take a portion of the culture, heat for 20 minutes at 100 ° C, and centrifuge at 13,000 rpm for 5 minutes. The supernatant was taken. Molecular diagnostic method using MTB-ID (M & D, Wonju, Korea) was used to classify tuberculosis bacteria and non-TB tuberculosis. PRA Myco-ID (M & D, Wonju, Korea) was used to identify non-TB tuberculosis species [Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000; 38: 2966-71. PRA Myco-ID was performed according to the test method provided by the manufacturer, the test method is as follows. The extracted nucleic acid was subjected to double-polymerase chain reaction, and the amplification product of 360 bp was confirmed by electrophoresis. The amplification products were treated with Msp I enzyme and subjected to electrophoresis on 4% agarose gels. The nucleic acid fragments appearing were analyzed according to the mycobacterial and nocadia identification algorithm provided by the manufacturer. If the exact nucleic acid fragmentation pattern was not corrected by Msp I enzyme treatment, the nucleic acid fragment pattern obtained by treatment with additional restriction enzymes such as Hae III and Alu I was confirmed. [Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000; 38: 2966-71.
실시예Example 5. 5. REBAREBA MycoMyco -- IDID 의 수행Performance of
REBA Myco-ID를 수행하기 위해서 검체로부터 분리된 핵산을 biotin이 표지된 primer를 이용하여 다음과 같이 PCR을 수행하였다. Pre-denaturation 과정으로 94 ℃ 에서 5분간 반응 후, denaturation 과정으로 94 ℃ 30초와 annealing과 elongation과정으로 65 ℃에서 30초로 45회 시행하고 최종 72 ℃에서 7분간 실시하여 270 bp의 PCR 산물을 증폭하였다.In order to perform REBA Myco-ID, PCR was performed using nucleic acid-labeled primers for nucleic acid isolated from the specimen. After 5 minutes of reaction at 94 ℃ for pre-denaturation, 45 times at 94 ℃ for 30 seconds and 30 seconds at 65 ℃ for annealing and elongation as a denaturation process, and amplification of 270 bp PCR product at 72 ℃ for 7 minutes. It was.
증폭한 PCR 산물을 이용한 REBA Myco-ID는 제조자가 제시한 실험조건을 이용하여 시행하였으면 실험방법은 다음과 같다. PCR 산물에 동량의 Denaturation solution (0.2N NaOH, 0.2mM EDTA)을 섞어 실온에 5분간 방치한 후 2X SSPE/0.1% SDS으로 희석시켜 준비된 REBA Myco-ID strip에 넣은 후 50℃ 에서 30분간 반응시키고, WS (washing solution)을 이용하여 62℃에서 10분간 두 번 씻어준 후 1:2000 (v/v)으로 희석한 alkaline phosphatase-labeled streptavidin conjugate (Roche, Mannheim, Germany)을 처리하여 실온에서 30분간 반응시켰다. 반응이 끝난 strip을 TBS용액 (pH7.5)으로 실온에 1분간 2회 세척하고, alkaline phosphatase에 반응하는 발색용 기질 용액인 NBT/BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolylphosphate, toluidine salt, Roche, Germany)를 pH 9.5인 TBS 용액으로 1:50으로 희석하여 실온에서 strip에 5분간 반응시키면서 프로브에 결합한 PCR 산물을 검출하였다.REBA Myco-ID using the amplified PCR product was carried out using the experimental conditions suggested by the manufacturer. REBA Myco-ID prepared by mixing the same amount of Denaturation solution (0.2N NaOH, 0.2mM EDTA) in the PCR product and allowed to stand at room temperature for 5 minutes, diluted with 2X SSPE / 0.1% SDS. After the reaction, the mixture was reacted for 30 minutes at 50 ° C, washed twice at 62 ° C with WS (washing solution) for 10 minutes, and diluted with alkaline phosphatase-labeled streptavidin conjugate (Roche, diluted 1: 2000 (v / v)). Mannheim, Germany) was treated for 30 minutes at room temperature. The reaction strip was washed twice with TBS solution (pH7.5) for 1 minute at room temperature, and NBT / BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3), a substrate solution for coloring reaction, reacted with alkaline phosphatase. -indolylphosphate, toluidine salt, Roche, Germany) was diluted 1:50 with TBS solution of pH 9.5 and reacted to the strip for 5 minutes at room temperature to detect the PCR product bound to the probe.
상기 실시예의 결과를 하기에서 설명한다.The result of the said Example is demonstrated below.
다중 multiple 중합효소연쇄반응(multiplex PCR)인Polymerase chain reaction (multiplex PCR) MTBMTB -- IDID 를 이용한 배양 검체 Cultured samples DNADNA 의 결핵균과 Mycobacterium tuberculosis 비결핵Non-tuberculosis 항산균Antibiotic acid 감별 Discrimination
검체에서 REBA-Myco ID의 결과와 비교 및 확인하고자 동일한 직접 검체의 액체배양에서 분리된 총 148개의 배양검체 DNA를 이용하여 MTB-ID (M&D, Korea)검사를 시행한 결과, 결핵균 59개의 검체와 비결핵 항산균 89개의 검체로 각각 분리되어 나왔으며, 도말 결과에 따라 분리를 해 본 결과 trace에서는 결핵균 4검체와 비결핵 항산균 5검체, 1+에서는 결핵균 23검체와 비결핵 항산균 22검체, 2+에서는 결핵균 11검체와 비결핵 항산균 31검체, 3+에서는 결핵균 6검체와 비결핵 항산균 14검체, 4+에서는 결핵균 15검체와 비결핵 항산균 17검체로 각각 검출된 것을 확인 할 수 있었다(표 1).
The MTB-ID (M & D, Korea) test was performed using DNA samples of 148 culture samples isolated from the liquid culture of the same direct sample to compare and confirm the results of REBA-Myco ID. 89 isolates of non-tuberculosis mycobacterium were isolated, and according to the results of the smear, traces showed 4 tuberculosis bacteria and 4 non-tuberculosis
REBAREBA MycoMyco -- IDID 를 이용한 객담 검체의 결핵균과 Mycobacterium tuberculosis 비결핵Non-tuberculosis 항산균Antibiotic acid 감별 Discrimination
객담 검체에서 분리된 DNA를 대상으로 하여 REBA-Myco ID를 수행하였다(도 1). 그 결과 결핵균 60검체와 비결핵 항산균 87검체로 각각 분리되어 나왔으며, 도말 결과에 따라 분리해 본 결과 trace에서 결핵균 4검체와 비결핵 항산균 5검체, 1+에서는 결핵균 24검체와 비결핵 항산균 21검체, 2+에서는 결핵균 11검체와 비결핵 항산균 31검체, 3+에서는 결핵균 6검체와 비결핵 항산균 14검체, 4+에서는 결핵균 15검체와 비결핵 항산균 16검체, 아무것도 확인되지 않은 1검체로 각각 검출 된 것을 확인 할 수 있었다(표 1).REBA-Myco ID was performed on DNA isolated from sputum specimens (FIG. 1). As a result, it was separated into 60 tuberculosis bacteria and non-tuberculosis mycobacterial 87 samples, respectively. According to the results of the smear, 4 tuberculosis bacteria and 5 non-tuberculosis acidophilic bacteria were tested in the trace, tuberculosis bacteria 24 and non-tuberculosis acidic acid in 1+. 21 specimens, 2+
객담에서 분리된 DNA를 이용한 REBA Myco-ID의 결과와 동일한 객담의 배양액에서 분리된 DNA를 이용한 MTB-ID의 결핵균과 비결핵 항산균 감별의 결과가 두 검체를 제외하고 모두 일치하였다. 결과가 일치하지 않은 도말 결과 1+인 한 검체에서는 MTB-ID의 결과상 비결핵 항산균으로 동정이 되었으나 REBA Myco-ID를 실시한 결과(도 2A)에서는 결핵균을 포함한 M. gordonae와 M. szulgai 의 비결핵 항산균이 섞여 나왔으며, 결핵균과 비결핵 항산균이 섞여 나온 이 검체의 경우 PRA Myco-ID결과는 제한효소에 의한 절편의 양상이 복합적으로 나타나서 균종을 판별할 수 없었다(표 3). 또한 다른 1개의 검체는 MTB-ID에서는 비결핵 항산균으로, REBA Myco-ID로는 어떠한 균종도 검출되지 않았다(도 2C). 이 검체의 배양액으로 PRA를 수행한 결과 Nocardia farcinia로 동정되었다(도 2D).
The results of the differentiation of M. tuberculosis and non-tuberculosis mycobacterium tuberculosis of MTB-ID using DNA isolated from the same sputum culture were identical with those of REBA Myco-ID. In one specimen with smear results, the result of MTB-ID was identified as non-tuberculosis mycobacterium, but REBA Myco-ID was performed (Fig. 2A), indicating that M. gordonae and M. szulgai , including tuberculosis, were found . Non-tuberculosis myco-IDs were mixed, and PRA Myco-ID resulted in a combination of restriction enzyme fragments, and the species could not be determined. In addition, the other sample was non-tuberculosis mycobacterium in MTB-ID, and no species was detected in REBA Myco-ID (Fig. 2C). Nocardia as a result of PRA with the culture of this sample It was identified as farcinia (FIG. 2D).
비결핵Non-tuberculosis 항산균의Antibacterial 검출분포 Detection Distribution
REBA-Myco ID를 통해 나온 비결핵항산균의 동정결과를 살펴본 결과 전체 87검체 중 M. intracellulare가 28검체(32.2%)로 가장 많이 동정이 되었으며, M. avium이 21검체(24.1%), M. massiliense가 19검체(21.8%), M. abscessus가 15검체(17.2%)의 순으로 분포하고 있었다. 그 외에 M. kansasii가 각각 1검체(1.2%)로 나왔으며, M. avium와 M. mucogenicum, M. avium와 M. abscessus, M. intracellulare와 M. scrofulaceum이 각각 섞여있는 경우가 1검체(1.2%)였다(Table 4). 또한, 두 종류의 이상의 비결핵 항산균이 섞여 나온 경우와 결핵균과 비결핵 항산균이 섞여나온 경우에 있어서 PRA Myco-ID 결과는 제한효소에 의한 절편의 양상이 섞여 있어서 균종을 판별할 수 없었다(도 2).REBA Myco-ID via the result of examining the result of identification of the non-tuberculosis mycobacteria out of the total 87 samples the M. intracellulare was identified as the most 28 specimens (32.2%), M. avium 21 specimens (24.1%), M . massiliense was the order of distribution of the 19 samples (21.8%), M. abscessus, 15 samples (17.2%). In addition the first sample (1.2 M. kansasii if each has a first or sample (1.2%), with M. avium and M. mucogenicum, M. avium and M. abscessus, M. intracellulare and M. scrofulaceum mixed with each %) (Table 4). In addition, in the case of mixing of two or more types of non-tuberculosis mycobacterium, and in the case of mycobacterium tuberculosis and non-tuberculosis mycobacterium, PRA Myco-ID results showed that the species of the fragments by restriction enzymes were mixed to determine the species. 2).
표 1은 객담을 사용한 REBA Myco-ID 및 배양을 사용한 MTB-ID에 의하여 결정된 및 한 MTB and NTM 분포를 나타낸 표이다.Table 1 is a table showing the MTB and NTM distribution as determined by REBA Myco-ID with sputum and MTB-ID with culture.
표 2는 다른 MTB 및 NTM 동정 테스트에 의한 결과 비교표이다.Table 2 is a comparison table of results by different MTB and NTM identification tests.
표 3는 REBA Myco-ID를 사용한 객담 DNA로부터 검출된 NTM 균주들의 분포를 나타낸 표이다.Table 3 is a table showing the distribution of NTM strains detected from sputum DNA using REBA Myco-ID.
표 4는 본 발명에 사용된 프라이머 및 프로브 서열을 나타낸 표이다. Table 4 is a table showing the primer and probe sequences used in the present invention.
표 5는 Sputum and Culture (SMC)를 사용하여 결정된 MTB/NTM 분포를 나타낸 표이다.
Table 5 shows the MTB / NTM distribution determined using Sputum and Culture (SMC).
<110> Molecules and Diagnostics Inc. <120> Method for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor <160> 29 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctgttcttc aaggagaagc gc 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acscggatct ggttctggat carc 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcaaggagaa gcgctacgac ctggc 25 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cgvcggttrc cgaagtggtc g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 5 gacgtcgtcg ccaccatcga 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 aagacrtcgt vgcsaccatc ga 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 tcggtctccc ggcgaactcc 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 8 catgtcggcg agcccatcac g 21 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 9 gccggtgagc cgatcacca 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 10 cggcctgcac gcgggcgag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 11 gcccgtacgg atggccagc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 12 aaaaggtgac caccaccacc 20 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 13 tgccggtgga cgtgg 15 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 14 aaatggtgac tgccaccacg 20 <210> 15 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 15 cctgaacgcc ggccag 16 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 16 aaaggccagc ccatcaccag c 21 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 17 cgtrccgggc ggggt 15 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 18 cgcttctcag gatggccagc 20 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 19 ccagccgacg atgacg 16 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 20 aaatgccggc cgcacccgcc gacgt 25 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 21 aaacctgacc gccaacccgg g 21 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 22 cgagagccca atcaccacc 19 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 23 cgcgtccccg atcacgac 18 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 24 aaagtcggcg atccgatca 19 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 25 tgaacgtcgg caagccga 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 26 aaagaacgtc ggcgagccg 19 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 27 ctgggcctgc acgacggcaa c 21 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 28 tcggtctccc ggcgaactcc 20 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 29 gtgccggtgg atgtg 15 <110> Molecules and Diagnostics Inc. <120> Method for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor <160> 29 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctgttcttc aaggagaagc gc 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acscggatct ggttctggat carc 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcaaggagaa gcgctacgac ctggc 25 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cgvcggttrc cgaagtggtc g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 5 gacgtcgtcg ccaccatcga 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 aagacrtcgt vgcsaccatc ga 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 tcggtctccc ggcgaactcc 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 8 catgtcggcg agcccatcac g 21 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 9 gccggtgagc cgatcacca 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 10 cggcctgcac gcgggcgag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 11 gcccgtacgg atggccagc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 12 aaaaggtgac caccaccacc 20 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 13 tgccggtgga cgtgg 15 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 14 aaatggtgac tgccaccacg 20 <210> 15 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 15 cctgaacgcc ggccag 16 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 16 aaaggccagc ccatcaccag c 21 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 17 cgtrccgggc ggggt 15 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 18 cgcttctcag gatggccagc 20 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 19 ccagccgacg atgacg 16 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 20 aaatgccggc cgcacccgcc gacgt 25 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 21 aaacctgacc gccaacccgg g 21 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 22 cgagagccca atcaccacc 19 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 23 cgcgtccccg atcacgac 18 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 24 aaagtcggcg atccgatca 19 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 25 tgaacgtcgg caagccga 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 26 aaagaacgtc ggcgagccg 19 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 27 ctgggcctgc acgacggcaa c 21 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 28 tcggtctccc ggcgaactcc 20 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 29 gtgccggtgg atgtg 15
Claims (9)
b)프라이머로서 서열번호 1 및 서열번호 2를 사용하여, 상기 DNA로부터 rpoB 유전자 상의 유전자 단편을 PCR 증폭하는 단계;및
c)서열번호 5 내지 29의 올리고머 프로브가 부착되어 있는 고체 서포트와 상기 단계 b)에서 얻어진 PCR 증폭산물을 하이브리드형성시키는 PCR-리버스 블랏 하이브리드형성 단계;
를 포함하는 것을 특징으로 하는 결핵균 및 비결핵 항산균의 구별 방법.a) separating DNA from a specimen sample;
b) PCR amplifying the gene fragment on the rpoB gene from said DNA using SEQ ID NO: 1 and SEQ ID NO: 2 as primers; and
c) a PCR-reverse blot hybridization step of hybridizing the solid support to which the oligomer probes of SEQ ID NOs: 5 to 29 are attached and the PCR amplification product obtained in step b);
Method for distinguishing Mycobacterium tuberculosis and non-tuberculosis antibacterial bacterium comprising a.
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